Protocol - Human Leukocyte Antigen (HLA) Genotyping - Assay
This protocol includes instructions for drawing, processing and storing blood according to the Type 1 Diabetes Genetics Consortium (T1DGC) Manual of Operations. Human Leukocyte Antigen is measured by using the linear array genotyping system method.
The PhenX Infectious Diseases and Immunity Working Group would like to note that the science of HLA matching is rapidly advancing and evolving and multiple methods for HLA assays are available.
To aid in comparability, the Infectious Diseases and Immunity Working Group recommends that the investigator record the make and manufacturer of equipment used and the repeatability and coefficients of variation for the assay.
Please also note that the immobilized linear arrays used to genotype the samples were provided by Roche Molecular Systems and are not commercially available.
The protocol for blood collection and processing is found in the Type 1 Diabetes Genetics Consortium Blood Collection and Processing Manual of Operations (T1DGC Manual of Operations). The protocol for the extraction of DNA from whole blood is found in Rosinger et al. 2010. The protocol for human leukocyte antigen genotyping is found in Mychaleckyj et al 2010. Please note that the immobilized linear arrays used to genotype the samples were provided by Roche Molecular Systems and are not commercially available.
The following is a summary version of the full Type 1 Diabetes Genetics Consortium protocols. It is not intended to replace the actual protocols listed above.
Venipuncture / Blood Collection Procedures
Editors Note: Please review chapter VI of the Type 1 Diabetes Genetics Consortium Blood Collection and Processing Manual of Operations (T1DGC Manual of Operations - Chapter VI) for a full description of Phlebotomy procedures.
- Blood is collected from the best available vein.
- Blood for DNA analysis should be collected in a 4.9-mL purple top tube (EDTA)
- The tubes are handled in a way that prevents hemolysis
- The tube contents are mixed by inverting eight times
- Record date and time of blood collection
- Record the reason why a blood sample was not collected.
Editors Note: Please review chapter VI of the Type 1 Diabetes Genetics Consortium Blood Collection and Processing Manual of Operations (T1DGC Manual of Operations - Chapter VI) for a full description of Blood Processing procedures.
- The sample is placed in an ice and water bath. Incubate the sample between 30 and 60 minutes.
- Do not let any of the samples stand in direct sunlight or at extreme temperatures.
- The sample is centrifuged, to separate the plasma from the cells.
- The plasma is pipetted away from the sample without disturbing the cell pack.
- The cell pack is shipped at ambient temperature to the DNA repository for DNA extraction.
DNA Extraction from Whole Blood
Please review Rosinger et al., 2010 for the full description of the DNA extraction protocol.
- DNA is isolated by a modified salting out procedure or by cholorform extraction
- DNA concentration is determined by fluorescence using a double-stranded DNA quantification reagent.
- DNA quality is confirmed by comparison to an appropriate standard (ladder) on an agarose gel.
Laboratory Assay for Human Leukocyte Antigen
Please review Mychaleckyj et al., 2010 for a full description of the HLA genotyping methods. Please note that the immobilized linear arrays used to genotype the samples were provided by Roche Molecular Systems and are not commercially available.
- DNA (5 ug total: 250 ul at 20ng/uL) is shipped to the HLA genotyping laboratory in screw top tubes.
- Each human leukocyte antigen region is amplified from 60 ng of genomic DNA by polymerase chain reaction (PCR) on a separate 96 well plate according to standardized protocol (see Mychaleckyj et al., 2010 for details of the polymerase chain reaction reaction)
- Polymerase chain reaction products are hybridized to oligonucleotide probes attached to nylon-backed membranes. The probes correspond to specific human leukocyte antigen DNA sequences. (Please note that the immobilized linear arrays used to genotype the samples were provided by Roche Molecular Systems and are not commercially available.)
- Hybridized probes are visualized and assigned a genotype by software.
Protocol Name from Source:
Personnel and Training Required
Laboratory capable of performing linear array genotyping
|Specialized requirements for biospecimen collection||No|
|Average time of greater than 15 minutes in an unaffected individual||No|
Mode of Administration
Toddler, Child, Adult
Children and Adults, 1 year and older.
The Type 1 Diabetes Genetic Consortium protocol was selected as the best standardized methodology for blood collection, processing and storage for the human leukocyte antigen genotyping assay. This protocol was used to genotype HLA-A, B, C, DRB1, DQ and DP loci in over 15,000 study participants between four different laboratory over five years.
|Common Data Elements (CDE)||HLA-A Gene Genotyping Allele Count||2693490||CDE Browser|
|Common Data Elements (CDE)||HLA-B Gene Genotyping Allele Count||2693496||CDE Browser|
|Common Data Elements (CDE)||HLA-C Gene Genotyping Allele Count||2693498||CDE Browser|
|Common Data Elements (CDE)||HLA-DRB1 Gene Genotyping Allele Count||2693500||CDE Browser|
|Common Data Elements (CDE)||HLA-DQB1 Gene Genotyping Allele Count||2693516||CDE Browser|
|Common Data Elements (CDE)||HLA-DPB1 Gene Genotyping Allele Count||2693520||CDE Browser|
|Common Data Elements (CDE)||HLA-DRB4 Gene Genotyping Allele Count||2704113||CDE Browser|
|Common Data Elements (CDE)||HLA-DRB5 Gene Genotyping Allele Count||2704130||CDE Browser|
|Common Data Elements (CDE)||HLA-DQA1 Gene Genotyping Allele Count||2704165||CDE Browser|
|Common Data Elements (CDE)||HLA-DPA1 Gene Genotyping Allele Count||2704192||CDE Browser|
|Common Data Elements (CDE)||HLA-DRB3 Gene Genotyping Allele Count||2705042||CDE Browser|
|Logical Observation Identifiers Names and Codes (LOINC)||Assay human leukocyte antg (HLA) proto||62875-0||LOINC|
|Human Phenotype Ontology||Abnormal leukocyte count||HP:0001881||HPO|
Process and Review
NIH. NIDDK. Type 1 Diabetes Genetics Consortium Manual Of Operations. Blood Collection and Processing.
Silke Rosinger, Sarah Nutland, Eric Mickelson, Michael D Varney, Bernard O Boehm, Gary J Olsem, John A Hansen, Ian Nicholson, Joan E Hilner, Letitia H Perdue, June J Pierce, Beena Akolkar, Concepcion Nierras, Michael W Steffes and T1DGC. Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA: the Type 1 Diabetes Genetics Consortium. Clinical Trials. 2010; 7: S65-S74.
Josyf C Mychaleckyj, Janelle A Noble, Priscilla V Moonsamy, Joyce A Carlson, Michael D Varner, Jeff Post, Wolfgang Helmberg, June J Pierce, Persia Bonella, Anna Lisa Fear, Eva Lavant, Anthony Louey, Sean Boyle, Julie A Lane, Paul Sali, Samuel Kim, Rebecca Rappner, Dustin T Williams, Letitia H Perdue, David M Reboussin, Brian D Tait, Beena Akolkar, Joan E Hilner, Michael W Steffes, Henry A Erlich and T1DGC. HLA genotyping in the international Type 1 Diabetes Genetics Consortium. Clinical Trials. 2010; 7: S75-S87.
|Variable Name||Variable ID||Variable Description||dbGaP Mapping|
|PX160601090000||Repeatability of the assay||N/A|
|PX160601030200||Record any comments about the blood draw, more||Variable Mapping|
|PX160601030000||Was blood drawn?||Variable Mapping|
|PX160601030100||Was full amount obtained?||Variable Mapping|
|PX160601100000||Coefficient of variation for the assay||N/A|
|PX160601010200||Date of blood Collection - day||N/A|
|PX160601010100||Date of blood Collection - month||N/A|
|PX160601010300||Date of blood Collection - year||N/A|
|PX160601060000||Enter comments to describe DNA quality||N/A|
|PX160601080100||Make of the equipment used to perform the more||N/A|
|PX160601080200||Manufacturer of the equipment used to more||N/A|
|PX160601110000||Results of the genotyping assay||N/A|
|PX160601070000||Record the type of assay used for HLA testing.||N/A|
|PX160601040000||Record any comments about the sample during more||Variable Mapping|
|PX160601020300||Time of blood collection - am or pm||N/A|
|PX160601020100||Time of blood collection - hour||N/A|
|PX160601020200||Time of blood collection - minutes||N/A|
Human Leukocyte Antigen (HLA) Genotyping Assay
November 12, 2010
A bioassay to obtain the specific allelic genotypes of several of the human leukocyte antigens, whose genes reside on chromosome 6p within the Major Histocompatibility Region (MHC).
The measure determines the alleles/single nucleotide polymorphisms that are present in the human leukocyte antigen regions of chromosome 6 to assess the major histocompatability system. The genetic variation within the human leukocyte antigen region determines the person's immune response and has been tied to specific autoimmune disorders such as Graves' disease and Hashimoto's thyroiditis, as well as other diseases such as multiple sclerosis, ankylosing spondylitis, and type 1 diabetes. This assay is similar to the genotyping performed in genome wide association studies but captures much more of the genetic variation within the human leukocyte antigen region than would be covered with "tag" single nucleotide polymorphisms.
Infectious disease, Human leukocyte antigen, HLA, Type 1 Diabetes Genetics Consortium, Infectious Diseases and Immunity